AIM: To investigate the roles and interactions of rhoassociatedprotein kinase (ROCK)1 and miR-124 inhuman colorectal cancer (CRC).METHODS: Expression of ROCK1 protein wasexamined by Western blotting, and quantitativer...AIM: To investigate the roles and interactions of rhoassociatedprotein kinase (ROCK)1 and miR-124 inhuman colorectal cancer (CRC).METHODS: Expression of ROCK1 protein wasexamined by Western blotting, and quantitativereverse transcriptase PCR was performed to measureexpression of ROCK1 mRNA and miR-124. Two cancercell lines were transfected with pre-miR-124 (mimic)and anti-miR-124 (inhibitor) and the effects onROCK1 protein and mRNA expression were observed.In addition, cell proliferation was assessed via a5-ethynyl-2′ deoxyuridine assay. Soft agar formationassay, and cell migration and invasion assays wereused to determine the effect of survivin on thetransformation and invasion activity of CRC cells.RESULTS: miR-124 was significantly downregulated inCRC compared to normal specimens (0.603 ± 0.092 vs1.147 ± 0.286, P = 0.016) and in metastatic comparedto nonmetastatic CRC specimens (0.416 ± 0.047 vs0.696 ± 0.089, P = 0.020). Expression of miR-124 wassignificantly associated with CRC metastasis, tumor Tand N stages, and tumor grade (all P < 0.05). ROCK1protein was significantly increased in CRC comparedto normal tissues (1.896 ± 0.258 vs 0.866 ± 0.136,P = 0.026), whereas ROCK1 mRNA expression wasunaltered (2.613 ± 0.251 vs 2.325 ± 0.246). miR-124and ROCK1 were inversely expressed in CRC tissuesand cell lines. ROCK1 mRNA was unaltered in cellstransfected with miR-124 mimic and miR-124 inhibitor,compared to normal controls. There was a significantreduction in ROCK1 protein in cells transfected withmiR-124 mimic and a significant increase in cells transfected with miR-124 inhibitor (P s < 0.05).Transformation and invasion of cells transfectedwith miR-124 inhibitor were significantly increasedcompared to those in normal controls (P < 0.05). Cellstransfected with miR-124 inhibitor showed increasedcell proliferation.CONCLUSION: miR-124 promotes hyperplasia andcontributes to invasion of CRC cells, but downregulatesROCK1. ROCK1 and miR-124 may play important rolesin CRC.展开更多
Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distri- bution of adhesive ...Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distri- bution of adhesive proteins to mediate neurite outgrowth remains unclear. By specifically modulat- ing Rho kinase activity with pharmacological agents, we studied the morpho-dynamics of neurite outgrowth. We found that lysophosphatidic acid, an activator of Rho kinase, inhibited neurite out- growth, which could be reversed by Y-27632, an inhibitor of Rho kinase. Meanwhile, reorganization of microtubules was noticed during these processes, as indicated by their significant changes in the soma and growth cone. In addition, exposure to lysophosphatidic acid led to a decreased mem- brane distribution of vinculin, a focal adhesion protein in neurons, whereas Y-27632 recruited vin- culin to the membrane. Taken together, our data suggest that Rho kinase regulates rat hippocampal neurite growth and microtubule formation via a mechanism associated with the redistribution of vinculin.展开更多
BACKGROUND Trastuzumab constitutes the fundamental component of initial therapy for patients with advanced human epidermal growth factor receptor 2(HER-2)-positive gastric cancer(GC).However,the efficacy of this treat...BACKGROUND Trastuzumab constitutes the fundamental component of initial therapy for patients with advanced human epidermal growth factor receptor 2(HER-2)-positive gastric cancer(GC).However,the efficacy of this treatment is hindered by substantial challenges associated with both primary and acquired drug resistance.While S-phase kinase associated protein 2(Skp2)overexpression has been implicated in the malignant progression of GC,its role in regulating trastuzumab resistance in this context remains uncertain.Despite the numerous studies investigating Skp2 inhibitors among small molecule compounds and natural products,there has been a lack of successful commercialization of drugs specifically targeting Skp2.AIM To discover a Skp2 blocker among currently available medications and develop a therapeutic strategy for HER2-positive GC patients who have experienced progression following trastuzumab-based treatment.METHODS Skp2 exogenous overexpression plasmids and small interfering RNA vectors were utilized to investigate the correlation between Skp2 expression and trastuzumab resistance in GC cells.Q-PCR,western blot,and immunohistochemical analyses were conducted to evaluate the regulatory effect of thioridazine on Skp2 expression.A cell counting kit-8 assay,flow cytometry,a amplex red glucose/glucose oxidase assay kit,and a lactate assay kit were utilized to measure the proliferation,apoptosis,and glycolytic activity of GC cells in vitro.A xenograft model established with human GC in nude mice was used to assess thioridazine's effectiveness in vivo.RESULTS The expression of Skp2 exhibited a negative correlation with the sensitivity of HER2-positive GC cells to trastuzumab.Thioridazine demonstrated the ability to directly bind to Skp2,resulting in a reduction in Skp2 expression at both the transcriptional and translational levels.Moreover,thioridazine effectively inhibited cell proliferation,exhibited antiapoptotic properties,and decreased the glucose uptake rate and lactate production by suppressing Skp2/protein kinase B/mammalian target of rapamycin/glucose transporter type 1 signaling pathways.The combination of thioridazine with either trastuzumab or lapatinib exhibited a more pronounced anticancer effect in vivo,surpassing the efficacy of either monotherapy.CONCLUSION Thioridazine demonstrates promising outcomes in preclinical GC models and offers a novel therapeutic approach for addressing trastuzumab resistance,particularly when used in conjunction with lapatinib.This compound has potential benefits for patients with Skp2-proficient tumors.展开更多
In this study, PC12 Adh cells and Neuro-2a cells were treated with Rho-associated kinase inhibitors （Y27632 and Fasudil）, a cyclooxygenase-1 selective inhibitor （SC560）, and a cyclooxygenase-2 inhibitor （NS398）....In this study, PC12 Adh cells and Neuro-2a cells were treated with Rho-associated kinase inhibitors （Y27632 and Fasudil）, a cyclooxygenase-1 selective inhibitor （SC560）, and a cyclooxygenase-2 inhibitor （NS398）. We found that these cells became tolerant to Rho-associated kinase inhibitors, as neurite outgrowth induced by these inhibitors diminished following more than 3 days of exposure in either cell line. The proteins cyclooxygenase-2 and cytosolic prostaglandin E synthetase were upregulated at day 3. NS398 decreased the tolerance to neurite outgrowth induction in both cell lines, whereas SC560 had almost no effect. These findings indicate that cells become tolerant to neurite outgrowth induced by Rho-associated kinase inhibitors, this is at least partly associated with upregulation of proteins involved in the cyclooxygenase-2 pathway, and cyclooxygenases-2 inhibition prevents this tolerance.展开更多
AIM:To investigate the role of Rho-associated protein kinase (ROCK) inhibitor, Y27632, in mediating the production of extracellular matrix (ECM) components including fibronectin, matrix metallo-proteinase-2 (MMP-2) an...AIM:To investigate the role of Rho-associated protein kinase (ROCK) inhibitor, Y27632, in mediating the production of extracellular matrix (ECM) components including fibronectin, matrix metallo-proteinase-2 (MMP-2) and type I collagen as induced by connective tissue growth factor(CTGF) or transforming growth factor-β (TGF-β) in a human retinal pigment epithelial cell line, ARPE-19. METHODS:The effect of Y27632 on the CTGF or TGF-β induced phenotype in ARPE-19 cells was measured with immunocytochemistry as the change in F-actin. ARPE-19 cells were treated with CTGF (1, 10, 100ng/mL)and TGF-β (10ng/mL) in serum free media, and analyzed for fibronectin, laminin, and MMP-2 and type I collagen by RT-qPCR and immunocytochemistry. Cells were also pretreated with an ROCK inhibitor, Y27632, to analyze the signaling contributing to ECM production. ·RESULTS:Treatment of ARPE-19 cells in culture with TGF-β or CTGF induced an ECM change from a cobblestone morphology to a more elongated swirl pattern indicating a mesenchymal phenotype. RT-qPCR analysis and different gene expression analysis demonstrated an upregulation in expression of genes associated with cytoskeletal structure and motility. CTGFor TGF-β significantly increased expression of fibronectin mRNA (P =0.006, P =0.003 respectively), laminin mRNA (P =0.006, P =0.005), MMP-2 mRNA (P =0.006, P =0.001), COL1A1 mRNA (P =0.001, P =0.001), COL1A2 mRNA (P = 0.001, P =0.001). Preincubation of ARPE-19 with Y27632 (10mmol/L) significantly prevented CTGF or TGF-β induced fibronectin (P=0.005, P=0.003 respectively), MMP-2 (P = 0.003, P =0.002), COL1A1 (P =0.006, P =0.003), and COL1A2 (P =0.006, P =0.004) gene expression, but not laminin (P =0.375, P =0.516). CONCLUSION:Our study demonstrated that both TGF-β and CTGF upregulate the expression of ECM components including fibronectin, laminin, MMP-2 and type I collagen by activating the RhoA/ROCK signaling pathway. During this process, ARPE-19 cells were shown to change from an epithelial to a mesenchymal phenotype in vitro. Y27632, a ROCK inhibitor, inhibited the transcription of fibronectin, MMP-2 and type I collagen, but not laminin. The data from our work suggest a role for CTGF as a profibrotic mediator. Inhibiting the RhoA/ROCK pathway represents a potential target to prevent the fibrosis of retinal pigment epithelial (RPE) cells. This might lead to a novel therapeutic approach to preventing the onset of early proliferative vitreoretinopathy(PVR).展开更多
Rho kinase inhibitor fasudil hydrochloride has been shown to reduce cerebral vasospasm, to inhibit inflammation and apoptosis and to promote the recovery of neurological function. However, the effect of fasudil hydroc...Rho kinase inhibitor fasudil hydrochloride has been shown to reduce cerebral vasospasm, to inhibit inflammation and apoptosis and to promote the recovery of neurological function. However, the effect of fasudil hydrochloride on claudin-5 protein expression has not been reported after cerebral ischemia/reperfusion. Therefore, this study sought to explore the effects of fasudil hydrochloride on blood-brain barrier permeability, growth-associated protein-43 and claudin-5 protein expression, and to further understand the neuroprotective effect of fasudil hydrochloride. A focal cerebral ischemia/reperfusion model was established using the intraluminal suture technique. Fasudil hydrochloride （15 mg/kg） was intraperitoneally injected once a day. Neurological deficit was evaluated using Longa＇s method. Changes in permeability of blood-brain barrier were measured using Evans blue. Changes in RhoA, growth-associated protein-43 and claudin-5 protein expression were detected using immunohistochemistry and western blotting. Results revealed that fasudil hydrochloride noticeably contributed to the recovery of neurological function, improved the function of blood-brain barrier, inhibited RhoA protein expression, and upregulated growth-associated protein-43 and claudin-5 protein expression following cerebral ischemia/reperfusion. Results indicated that Rho kinase exhibits a certain effect on neurovascular damage following cerebral ischemia/reperfusion. Intervention targeted Rho kinase might be a new therapeutic target in the treatment of cerebral ischemia/reperfusion.展开更多
目的探讨Rho激酶(ROCK)在氢气改善离体脓毒症肠屏障功能中的作用。方法常规培养人结肠上皮细胞Caco-2,分为6组(n=3):对照组(C组)、富氢培养基组(H组)、脂多糖(LPS)处理组(L组)、富氢培养基+LPS组(HL组)、Rho激酶抑制剂Y-27632组(Y组)、Y...目的探讨Rho激酶(ROCK)在氢气改善离体脓毒症肠屏障功能中的作用。方法常规培养人结肠上皮细胞Caco-2,分为6组(n=3):对照组(C组)、富氢培养基组(H组)、脂多糖(LPS)处理组(L组)、富氢培养基+LPS组(HL组)、Rho激酶抑制剂Y-27632组(Y组)、Y-27632+LPS组(YL组)。H组给予0.6 mmol/L富氢培养基;LPS和Y-27632的处理浓度分别为50 mg/L、25μmol/L。建立Transwell小室模型,定期检测跨上皮电阻值(TEER值),当TEER值达到800Ω·cm^2后给予处理,于6、12、24 h检测TEER值,24 h时检测FITC-右旋糖酐通过率。细胞接种于6孔板,融合达80%~90%后给予处理,实时聚合酶链式反应技术检测闭锁小带蛋白1(ZO-1)mRNA和ROCK mRNA表达情况;蛋白免疫印迹技术检测ZO-1蛋白和ROCK蛋白表达水平。结果与C组比较,H组12、24 h TEER值升高(P<0.05),FITC-右旋糖酐通过率、ZO-1蛋白和ROCK蛋白表达水平差异无统计学意义;Y组6、12、24 h TEER值升高(P<0.05),FITC-右旋糖酐通过率差异无统计学意义,ZO-1 mRNA表达增加,ROCK mRNA表达减少(均P<0.05);L组6、12、24 h TEER值降低,FITC-右旋糖酐通过率增高,ZO-1 mRNA和蛋白表达均下降,ROCK mRNA和蛋白表达均增加(P<0.05)。与L组比较,6、12、24 h YL组TEER值增高,FITC-右旋糖酐通过率降低,ZO-1 mRNA表达增加,ROCK mRNA表达降低(均P<0.05)。与L组比较,HL组6、12、24 h TEER值增高,FITC-右旋糖酐通过率降低,各时间点ZO-1蛋白表达上升,ROCK蛋白表达下降(均P<0.05)。结论氢气可保护脓毒症肠屏障功能,改善肠上皮屏障完整性和通透性,增加肠细胞间紧密连接蛋白表达,这些保护机制可能与氢气抑制LPS诱导的ROCK过度表达有关。展开更多
目的:观察Rho激酶抑制剂法舒地尔(Fasudil)对人高侵袭潜能HCC细胞株3(human high metastatic liver cancer cells 3,HCCLM3)侵袭转移的影响,并且探讨其作用的机制。方法:应用100 mol/L Fasudil作用于HCCLM3细胞,采用肌动蛋白微丝荧光染...目的:观察Rho激酶抑制剂法舒地尔(Fasudil)对人高侵袭潜能HCC细胞株3(human high metastatic liver cancer cells 3,HCCLM3)侵袭转移的影响,并且探讨其作用的机制。方法:应用100 mol/L Fasudil作用于HCCLM3细胞,采用肌动蛋白微丝荧光染色和侵袭小室实验观察HCCLM3细胞的运动侵袭能力。HCCLM3细胞经过处理后分为阴性对照组、Fasudil作用组、BTBD7干扰组,通过Western印迹检测BTB/POZ结构域蛋白7(BR-C,ttk and bab/pox virus domain containing 7,BTBD7)、Ras同系物家族成员C(ras homolog family member C,Rho C)、Rho关联卷曲螺旋蛋白激酶2(Rhoassociated,coiled-coil containing protein kinase 2,ROCK2)、MMP2和MMP9蛋白表达水平,酶谱分析法检测MMP2和MMP9活性水平。BTBD7干扰组作为阳性对照。结果:Fasudil处理后HCCLM3侵袭运动能力下降,BTBD7,Rho C,ROCK2蛋白表达下调,MMP2和MMP9活性降低,与阴性对照组比较差异有统计学意义(均P<0.01)。结论:Fasudil具有干预BTBD7-ROCK2信号通路、抑制HCC侵袭转移的重要作用。展开更多
目的研究Rho相关卷曲螺旋蛋白激酶1(ROCK1)在动脉粥样硬化血管壁中的表达及其与基质金属蛋白酶2(MMP2)、转化生长因子1(TGF-β1)的相关性。方法选择30只载脂蛋白E基因敲除小鼠为实验组,高脂饲料喂养,另选30只C57BL/6小鼠为对照组,普通...目的研究Rho相关卷曲螺旋蛋白激酶1(ROCK1)在动脉粥样硬化血管壁中的表达及其与基质金属蛋白酶2(MMP2)、转化生长因子1(TGF-β1)的相关性。方法选择30只载脂蛋白E基因敲除小鼠为实验组,高脂饲料喂养,另选30只C57BL/6小鼠为对照组,普通饲料喂养。在喂养的第10、16、22、28及34周,取眼球血监测小鼠血脂水平;取小鼠腹主动脉作为标本,包埋切片并进行苏木精-伊红染色观察血管壁形态;应用免疫组化染色观察血管壁中ROCK1、MMP2、TGF-β1的表达;使用Image Pro Plus 6.0软件测量切片中血管壁厚度、斑块面积、血管壁中ROCK1、MMP2及TGF-β1的表达量。采用SPSS 27.0统计软件进行数据分析。采用单因素方差分析进行组间比较,两两比较采用Tukey检验。采用Pearson相关与线性回归分析ROCK1与血管壁厚度及斑块面积、MMP2、TGF-β1的关系。结果成功建立动脉粥样硬化小鼠模型。在喂养第10、16、22、28及34周,实验组血脂水平明显高于对照组,差异有统计学意义(P<0.05)。自喂养第16周起,实验组小鼠血管内均有斑块形成,随着喂养时间的延长,斑块面积和血管壁厚度不断增加,差异有统计学意义(P<0.05);血管壁内ROCK1的表达逐步增高,其表达与斑块面积及血管壁厚度呈正相关(r=0.821,0.730;P<0.05)。线性相关分析及回归分析显示,ROCK1与MMP2、TGF-β1表达均呈正相关(r=0.801,0.906;P<0.05)。结论在动脉粥样硬化血管壁中存在ROCK1蛋白的表达,且表达量随着血管壁厚度增加而增高,ROCK1的表达与MMP2、TGF-β1呈显著正相关性。鉴于ROCK1蛋白的致血管痉挛作用,提示动脉粥样硬化血管壁可能易于痉挛,具体机制需进一步研究。展开更多
BACKGROUND: The increased β-arrestin-2 and its combination with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric ...BACKGROUND: The increased β-arrestin-2 and its combination with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric arteries of cirrhotic patients and rats. The present study is to investigate the machinery changes of α-adrenergic receptors and G proteins and their roles in the contractility of mesenteric arteries of cirrhotic patients and animal models. METHODS: Patients with cirrhosis due to hepatitis B and cirrhotic rats induced by CCl 4 were studied. Mesenteric artery contractility in response to norepinephrine was determined by a vessel perfusion system. The contractile effect of G protein-coupled receptor kinase-2 (GRK-2) inhibitor on the mesenteric artery was evaluated. The protein expression of the α 1 adrenergic receptor, G proteins, β-arrestin-2, GRK-2 as well as the activity of Rho associated coiled-coil forming protein kinase-1 (ROCK-1) were measured by Western blot. In addition, the interaction of α 1 adrenergic receptor with β-arrestin-2 was assessed by co-immunoprecipitation. RESULTS: The portal vein pressure of cirrhotic patients and rats was significantly higher than that of controls. The doseresponse curve to norepinephrine in mesenteric arteriole was shifted to the right, and EC 50 was significantly increased in cirrhotic patients and rats. There were no significant differences in the expressions of the α 1 adrenergic receptor and G proteins in the cirrhotic group compared with the controls. However, the protein expressions of GRK-2 and β-arrestin-2 were significantly elevated in cirrhotic patients and rats compared with those of the controls. The interaction of the α 1 adrenergic receptor and β-arrestin-2 was significantly aggravated. This interaction was significantly reversed by GRK-2 inhibitor. Both the protein expression and activity of ROCK-1 were significantly decreased in the mesenteric artery in patients with cirrhosis compared with those of the controls, and this phenomenon was not shown in the cirrhotic rats. Norepinephrine significantly increased the activity of ROCK-1 in normal rats but not in cirrhotic ones. Norepinephrine significantly increased ROCK-1 activity in cirrhotic rats when GRK-2 inhibitor was used. CONCLUSIONS: β-arrestin-2 expression and its interaction with GPCRs are significantly upregulated in the mesenteric arteries in patients and rats with cirrhosis. These upregulations result in GPCR desensitization, G-protein dysfunction and ROCK inhibition. These may explain the decreased contractility of the mesenteric artery in response to vasoconstrictors.展开更多
目的检测IgA肾病(immunoglobulin A nephropathy,IgAN)患者血清Rho相关卷曲螺旋形成蛋白激酶2(rho associated coiled coil containing protein kinase 2,ROCK2)、视黄醇结合蛋白4(retinol-binding protein 4,RBP4)表达水平,分析二者在...目的检测IgA肾病(immunoglobulin A nephropathy,IgAN)患者血清Rho相关卷曲螺旋形成蛋白激酶2(rho associated coiled coil containing protein kinase 2,ROCK2)、视黄醇结合蛋白4(retinol-binding protein 4,RBP4)表达水平,分析二者在评估患者病情及预测预后中的价值。方法选取2016年6月至2019年6月98例中国贵航集团302医院已确诊收治的IgAN患者为IgAN组,其中轻度IgAN 36例、中度IgAN 32例、重度IgAN 30例;同期选择在中国贵航集团302医院体检的健康者100名为对照组。酶联免疫吸附法检测血清中ROCK2、RBP4水平;采用Kaplan-Meier生存曲线分析ROCK2、RBP4表达水平与IgAN患者预后的关系;Pearson相关性分析ROCK2与RBP4表达相关性;受试者工作特征曲线(receiver operating characteristic curve,ROC)分析ROCK2、RBP4对IgAN的诊断价值;比例风险回归模型法分析IgAN患者预后的影响因素。结果IgAN组血清中ROCK2、RBP4表达水平分别为(24.25±5.31)μg/L、(59.70±9.43)μg/L,显著高于对照组的(15.57±4.16)μg/L、(32.14±7.82)μg/L,差异具有统计学意义(t=12.818、22.404,均P<0.001);轻度、中度、重度IgAN患者血清中ROCK2水平分别为(18.69±4.36)μg/L、(23.75±5.31)μg/L、(31.46±5.62)μg/L,RBP4表达水平分别为(45.37±7.86)μg/L、(62.48±8.12)μg/L、(73.94±8.65)μg/L。随着病情的加重,IgAN患者血清ROCK2、RBP4表达水平逐渐升高,差异具有统计学意义(F=51.854、102.234,均P<0.05);IgAN患者血清中ROCK2、RBP4表达水平与Lee氏分级、IgA/C3比值、病情程度有关(P<0.05);ROCK2、RBP4高表达组患者预后生存率分别为60.00%、74.19%,均显著低于低表达组的89.71%、91.67%,差异具有统计学意义(log rankχ^(2)=4.674,P=0.031);ROC曲线结果显示,血清ROCK2诊断IgAN的曲线下面积(area under curve,AUC)为0.889,敏感度为80.61%,特异性为80.00%,截断值为18.79μg/L;RBP4诊断IgAN的AUC为0.986,敏感度为96.94%,特异性为95.00%,截断值为41.31μg/L;二者联合检测的AUC为0.997,敏感度为98.98%,特异性为94.86%。Pearson相关性分析显示,IgAN患者血清ROCK2与RBP4表达水平呈显著正相关(r=0.701,P<0.001);多因素比例风险回归模型法分析表明,IgA/C3(OR=2.683,95%CI:1.060~6.794)、ROCK2(OR=1.831,95%CI:1.027~3.264)、RBP4(OR=1.517,95%CI:1.104~2.084)均是IgAN患者预后生存的影响因素(均P<0.05)。结论ROCK2、RBP4在IgAN患者血清中高表达,二者与IgAN患者的临床病理特征、病情严重程度及预后关系密切,ROCK2、RBP4表达水平对IgAN的诊断具有一定的价值。展开更多
文摘AIM: To investigate the roles and interactions of rhoassociatedprotein kinase (ROCK)1 and miR-124 inhuman colorectal cancer (CRC).METHODS: Expression of ROCK1 protein wasexamined by Western blotting, and quantitativereverse transcriptase PCR was performed to measureexpression of ROCK1 mRNA and miR-124. Two cancercell lines were transfected with pre-miR-124 (mimic)and anti-miR-124 (inhibitor) and the effects onROCK1 protein and mRNA expression were observed.In addition, cell proliferation was assessed via a5-ethynyl-2′ deoxyuridine assay. Soft agar formationassay, and cell migration and invasion assays wereused to determine the effect of survivin on thetransformation and invasion activity of CRC cells.RESULTS: miR-124 was significantly downregulated inCRC compared to normal specimens (0.603 ± 0.092 vs1.147 ± 0.286, P = 0.016) and in metastatic comparedto nonmetastatic CRC specimens (0.416 ± 0.047 vs0.696 ± 0.089, P = 0.020). Expression of miR-124 wassignificantly associated with CRC metastasis, tumor Tand N stages, and tumor grade (all P < 0.05). ROCK1protein was significantly increased in CRC comparedto normal tissues (1.896 ± 0.258 vs 0.866 ± 0.136,P = 0.026), whereas ROCK1 mRNA expression wasunaltered (2.613 ± 0.251 vs 2.325 ± 0.246). miR-124and ROCK1 were inversely expressed in CRC tissuesand cell lines. ROCK1 mRNA was unaltered in cellstransfected with miR-124 mimic and miR-124 inhibitor,compared to normal controls. There was a significantreduction in ROCK1 protein in cells transfected withmiR-124 mimic and a significant increase in cells transfected with miR-124 inhibitor (P s < 0.05).Transformation and invasion of cells transfectedwith miR-124 inhibitor were significantly increasedcompared to those in normal controls (P < 0.05). Cellstransfected with miR-124 inhibitor showed increasedcell proliferation.CONCLUSION: miR-124 promotes hyperplasia andcontributes to invasion of CRC cells, but downregulatesROCK1. ROCK1 and miR-124 may play important rolesin CRC.
基金supported by the National Natural Science Foundation of China,No.31170941the Fundamental Research Funds for the Central Universities,No.21612424the Science and Technology Planning Project of Guangdong Province,No.2010B031600102
文摘Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distri- bution of adhesive proteins to mediate neurite outgrowth remains unclear. By specifically modulat- ing Rho kinase activity with pharmacological agents, we studied the morpho-dynamics of neurite outgrowth. We found that lysophosphatidic acid, an activator of Rho kinase, inhibited neurite out- growth, which could be reversed by Y-27632, an inhibitor of Rho kinase. Meanwhile, reorganization of microtubules was noticed during these processes, as indicated by their significant changes in the soma and growth cone. In addition, exposure to lysophosphatidic acid led to a decreased mem- brane distribution of vinculin, a focal adhesion protein in neurons, whereas Y-27632 recruited vin- culin to the membrane. Taken together, our data suggest that Rho kinase regulates rat hippocampal neurite growth and microtubule formation via a mechanism associated with the redistribution of vinculin.
基金Supported by Youth Fund of National Natural Science Foundation of China,No.81803575,No.31902287Kaifeng Science and Technology Development Plan Project,No.2203008+2 种基金Key Specialized Research and Promotion Project of Henan Province in 2023,No.232102311205Henan Medical Science and Technology Research Program Project,No.LHGJ20210801College Students Innovation and Entrepreneurship Training Program of Henan University,No.20231022007.
文摘BACKGROUND Trastuzumab constitutes the fundamental component of initial therapy for patients with advanced human epidermal growth factor receptor 2(HER-2)-positive gastric cancer(GC).However,the efficacy of this treatment is hindered by substantial challenges associated with both primary and acquired drug resistance.While S-phase kinase associated protein 2(Skp2)overexpression has been implicated in the malignant progression of GC,its role in regulating trastuzumab resistance in this context remains uncertain.Despite the numerous studies investigating Skp2 inhibitors among small molecule compounds and natural products,there has been a lack of successful commercialization of drugs specifically targeting Skp2.AIM To discover a Skp2 blocker among currently available medications and develop a therapeutic strategy for HER2-positive GC patients who have experienced progression following trastuzumab-based treatment.METHODS Skp2 exogenous overexpression plasmids and small interfering RNA vectors were utilized to investigate the correlation between Skp2 expression and trastuzumab resistance in GC cells.Q-PCR,western blot,and immunohistochemical analyses were conducted to evaluate the regulatory effect of thioridazine on Skp2 expression.A cell counting kit-8 assay,flow cytometry,a amplex red glucose/glucose oxidase assay kit,and a lactate assay kit were utilized to measure the proliferation,apoptosis,and glycolytic activity of GC cells in vitro.A xenograft model established with human GC in nude mice was used to assess thioridazine's effectiveness in vivo.RESULTS The expression of Skp2 exhibited a negative correlation with the sensitivity of HER2-positive GC cells to trastuzumab.Thioridazine demonstrated the ability to directly bind to Skp2,resulting in a reduction in Skp2 expression at both the transcriptional and translational levels.Moreover,thioridazine effectively inhibited cell proliferation,exhibited antiapoptotic properties,and decreased the glucose uptake rate and lactate production by suppressing Skp2/protein kinase B/mammalian target of rapamycin/glucose transporter type 1 signaling pathways.The combination of thioridazine with either trastuzumab or lapatinib exhibited a more pronounced anticancer effect in vivo,surpassing the efficacy of either monotherapy.CONCLUSION Thioridazine demonstrates promising outcomes in preclinical GC models and offers a novel therapeutic approach for addressing trastuzumab resistance,particularly when used in conjunction with lapatinib.This compound has potential benefits for patients with Skp2-proficient tumors.
基金supported by Yunnan Provincial Science and Technology Department, No.2009CD079the National Natural Science Foundation ofChina, No.81060109.
文摘In this study, PC12 Adh cells and Neuro-2a cells were treated with Rho-associated kinase inhibitors （Y27632 and Fasudil）, a cyclooxygenase-1 selective inhibitor （SC560）, and a cyclooxygenase-2 inhibitor （NS398）. We found that these cells became tolerant to Rho-associated kinase inhibitors, as neurite outgrowth induced by these inhibitors diminished following more than 3 days of exposure in either cell line. The proteins cyclooxygenase-2 and cytosolic prostaglandin E synthetase were upregulated at day 3. NS398 decreased the tolerance to neurite outgrowth induction in both cell lines, whereas SC560 had almost no effect. These findings indicate that cells become tolerant to neurite outgrowth induced by Rho-associated kinase inhibitors, this is at least partly associated with upregulation of proteins involved in the cyclooxygenase-2 pathway, and cyclooxygenases-2 inhibition prevents this tolerance.
文摘AIM:To investigate the role of Rho-associated protein kinase (ROCK) inhibitor, Y27632, in mediating the production of extracellular matrix (ECM) components including fibronectin, matrix metallo-proteinase-2 (MMP-2) and type I collagen as induced by connective tissue growth factor(CTGF) or transforming growth factor-β (TGF-β) in a human retinal pigment epithelial cell line, ARPE-19. METHODS:The effect of Y27632 on the CTGF or TGF-β induced phenotype in ARPE-19 cells was measured with immunocytochemistry as the change in F-actin. ARPE-19 cells were treated with CTGF (1, 10, 100ng/mL)and TGF-β (10ng/mL) in serum free media, and analyzed for fibronectin, laminin, and MMP-2 and type I collagen by RT-qPCR and immunocytochemistry. Cells were also pretreated with an ROCK inhibitor, Y27632, to analyze the signaling contributing to ECM production. ·RESULTS:Treatment of ARPE-19 cells in culture with TGF-β or CTGF induced an ECM change from a cobblestone morphology to a more elongated swirl pattern indicating a mesenchymal phenotype. RT-qPCR analysis and different gene expression analysis demonstrated an upregulation in expression of genes associated with cytoskeletal structure and motility. CTGFor TGF-β significantly increased expression of fibronectin mRNA (P =0.006, P =0.003 respectively), laminin mRNA (P =0.006, P =0.005), MMP-2 mRNA (P =0.006, P =0.001), COL1A1 mRNA (P =0.001, P =0.001), COL1A2 mRNA (P = 0.001, P =0.001). Preincubation of ARPE-19 with Y27632 (10mmol/L) significantly prevented CTGF or TGF-β induced fibronectin (P=0.005, P=0.003 respectively), MMP-2 (P = 0.003, P =0.002), COL1A1 (P =0.006, P =0.003), and COL1A2 (P =0.006, P =0.004) gene expression, but not laminin (P =0.375, P =0.516). CONCLUSION:Our study demonstrated that both TGF-β and CTGF upregulate the expression of ECM components including fibronectin, laminin, MMP-2 and type I collagen by activating the RhoA/ROCK signaling pathway. During this process, ARPE-19 cells were shown to change from an epithelial to a mesenchymal phenotype in vitro. Y27632, a ROCK inhibitor, inhibited the transcription of fibronectin, MMP-2 and type I collagen, but not laminin. The data from our work suggest a role for CTGF as a profibrotic mediator. Inhibiting the RhoA/ROCK pathway represents a potential target to prevent the fibrosis of retinal pigment epithelial (RPE) cells. This might lead to a novel therapeutic approach to preventing the onset of early proliferative vitreoretinopathy(PVR).
基金funded by the National Natural Science Foundation of China,No.30870855the Natural Science Foundation of Beijing,No.7082028Beijing Municipal Health System High-Level Technician Cultivation Project,No.2009-3-07
文摘Rho kinase inhibitor fasudil hydrochloride has been shown to reduce cerebral vasospasm, to inhibit inflammation and apoptosis and to promote the recovery of neurological function. However, the effect of fasudil hydrochloride on claudin-5 protein expression has not been reported after cerebral ischemia/reperfusion. Therefore, this study sought to explore the effects of fasudil hydrochloride on blood-brain barrier permeability, growth-associated protein-43 and claudin-5 protein expression, and to further understand the neuroprotective effect of fasudil hydrochloride. A focal cerebral ischemia/reperfusion model was established using the intraluminal suture technique. Fasudil hydrochloride （15 mg/kg） was intraperitoneally injected once a day. Neurological deficit was evaluated using Longa＇s method. Changes in permeability of blood-brain barrier were measured using Evans blue. Changes in RhoA, growth-associated protein-43 and claudin-5 protein expression were detected using immunohistochemistry and western blotting. Results revealed that fasudil hydrochloride noticeably contributed to the recovery of neurological function, improved the function of blood-brain barrier, inhibited RhoA protein expression, and upregulated growth-associated protein-43 and claudin-5 protein expression following cerebral ischemia/reperfusion. Results indicated that Rho kinase exhibits a certain effect on neurovascular damage following cerebral ischemia/reperfusion. Intervention targeted Rho kinase might be a new therapeutic target in the treatment of cerebral ischemia/reperfusion.
文摘目的探讨Rho激酶(ROCK)在氢气改善离体脓毒症肠屏障功能中的作用。方法常规培养人结肠上皮细胞Caco-2,分为6组(n=3):对照组(C组)、富氢培养基组(H组)、脂多糖(LPS)处理组(L组)、富氢培养基+LPS组(HL组)、Rho激酶抑制剂Y-27632组(Y组)、Y-27632+LPS组(YL组)。H组给予0.6 mmol/L富氢培养基;LPS和Y-27632的处理浓度分别为50 mg/L、25μmol/L。建立Transwell小室模型,定期检测跨上皮电阻值(TEER值),当TEER值达到800Ω·cm^2后给予处理,于6、12、24 h检测TEER值,24 h时检测FITC-右旋糖酐通过率。细胞接种于6孔板,融合达80%~90%后给予处理,实时聚合酶链式反应技术检测闭锁小带蛋白1(ZO-1)mRNA和ROCK mRNA表达情况;蛋白免疫印迹技术检测ZO-1蛋白和ROCK蛋白表达水平。结果与C组比较,H组12、24 h TEER值升高(P<0.05),FITC-右旋糖酐通过率、ZO-1蛋白和ROCK蛋白表达水平差异无统计学意义;Y组6、12、24 h TEER值升高(P<0.05),FITC-右旋糖酐通过率差异无统计学意义,ZO-1 mRNA表达增加,ROCK mRNA表达减少(均P<0.05);L组6、12、24 h TEER值降低,FITC-右旋糖酐通过率增高,ZO-1 mRNA和蛋白表达均下降,ROCK mRNA和蛋白表达均增加(P<0.05)。与L组比较,6、12、24 h YL组TEER值增高,FITC-右旋糖酐通过率降低,ZO-1 mRNA表达增加,ROCK mRNA表达降低(均P<0.05)。与L组比较,HL组6、12、24 h TEER值增高,FITC-右旋糖酐通过率降低,各时间点ZO-1蛋白表达上升,ROCK蛋白表达下降(均P<0.05)。结论氢气可保护脓毒症肠屏障功能,改善肠上皮屏障完整性和通透性,增加肠细胞间紧密连接蛋白表达,这些保护机制可能与氢气抑制LPS诱导的ROCK过度表达有关。
文摘目的:观察Rho激酶抑制剂法舒地尔(Fasudil)对人高侵袭潜能HCC细胞株3(human high metastatic liver cancer cells 3,HCCLM3)侵袭转移的影响,并且探讨其作用的机制。方法:应用100 mol/L Fasudil作用于HCCLM3细胞,采用肌动蛋白微丝荧光染色和侵袭小室实验观察HCCLM3细胞的运动侵袭能力。HCCLM3细胞经过处理后分为阴性对照组、Fasudil作用组、BTBD7干扰组,通过Western印迹检测BTB/POZ结构域蛋白7(BR-C,ttk and bab/pox virus domain containing 7,BTBD7)、Ras同系物家族成员C(ras homolog family member C,Rho C)、Rho关联卷曲螺旋蛋白激酶2(Rhoassociated,coiled-coil containing protein kinase 2,ROCK2)、MMP2和MMP9蛋白表达水平,酶谱分析法检测MMP2和MMP9活性水平。BTBD7干扰组作为阳性对照。结果:Fasudil处理后HCCLM3侵袭运动能力下降,BTBD7,Rho C,ROCK2蛋白表达下调,MMP2和MMP9活性降低,与阴性对照组比较差异有统计学意义(均P<0.01)。结论:Fasudil具有干预BTBD7-ROCK2信号通路、抑制HCC侵袭转移的重要作用。
文摘目的研究Rho相关卷曲螺旋蛋白激酶1(ROCK1)在动脉粥样硬化血管壁中的表达及其与基质金属蛋白酶2(MMP2)、转化生长因子1(TGF-β1)的相关性。方法选择30只载脂蛋白E基因敲除小鼠为实验组,高脂饲料喂养,另选30只C57BL/6小鼠为对照组,普通饲料喂养。在喂养的第10、16、22、28及34周,取眼球血监测小鼠血脂水平;取小鼠腹主动脉作为标本,包埋切片并进行苏木精-伊红染色观察血管壁形态;应用免疫组化染色观察血管壁中ROCK1、MMP2、TGF-β1的表达;使用Image Pro Plus 6.0软件测量切片中血管壁厚度、斑块面积、血管壁中ROCK1、MMP2及TGF-β1的表达量。采用SPSS 27.0统计软件进行数据分析。采用单因素方差分析进行组间比较,两两比较采用Tukey检验。采用Pearson相关与线性回归分析ROCK1与血管壁厚度及斑块面积、MMP2、TGF-β1的关系。结果成功建立动脉粥样硬化小鼠模型。在喂养第10、16、22、28及34周,实验组血脂水平明显高于对照组,差异有统计学意义(P<0.05)。自喂养第16周起,实验组小鼠血管内均有斑块形成,随着喂养时间的延长,斑块面积和血管壁厚度不断增加,差异有统计学意义(P<0.05);血管壁内ROCK1的表达逐步增高,其表达与斑块面积及血管壁厚度呈正相关(r=0.821,0.730;P<0.05)。线性相关分析及回归分析显示,ROCK1与MMP2、TGF-β1表达均呈正相关(r=0.801,0.906;P<0.05)。结论在动脉粥样硬化血管壁中存在ROCK1蛋白的表达,且表达量随着血管壁厚度增加而增高,ROCK1的表达与MMP2、TGF-β1呈显著正相关性。鉴于ROCK1蛋白的致血管痉挛作用,提示动脉粥样硬化血管壁可能易于痉挛,具体机制需进一步研究。
基金supported by a grant from the National Natural Science Foundation of China (30972920)
文摘BACKGROUND: The increased β-arrestin-2 and its combination with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric arteries of cirrhotic patients and rats. The present study is to investigate the machinery changes of α-adrenergic receptors and G proteins and their roles in the contractility of mesenteric arteries of cirrhotic patients and animal models. METHODS: Patients with cirrhosis due to hepatitis B and cirrhotic rats induced by CCl 4 were studied. Mesenteric artery contractility in response to norepinephrine was determined by a vessel perfusion system. The contractile effect of G protein-coupled receptor kinase-2 (GRK-2) inhibitor on the mesenteric artery was evaluated. The protein expression of the α 1 adrenergic receptor, G proteins, β-arrestin-2, GRK-2 as well as the activity of Rho associated coiled-coil forming protein kinase-1 (ROCK-1) were measured by Western blot. In addition, the interaction of α 1 adrenergic receptor with β-arrestin-2 was assessed by co-immunoprecipitation. RESULTS: The portal vein pressure of cirrhotic patients and rats was significantly higher than that of controls. The doseresponse curve to norepinephrine in mesenteric arteriole was shifted to the right, and EC 50 was significantly increased in cirrhotic patients and rats. There were no significant differences in the expressions of the α 1 adrenergic receptor and G proteins in the cirrhotic group compared with the controls. However, the protein expressions of GRK-2 and β-arrestin-2 were significantly elevated in cirrhotic patients and rats compared with those of the controls. The interaction of the α 1 adrenergic receptor and β-arrestin-2 was significantly aggravated. This interaction was significantly reversed by GRK-2 inhibitor. Both the protein expression and activity of ROCK-1 were significantly decreased in the mesenteric artery in patients with cirrhosis compared with those of the controls, and this phenomenon was not shown in the cirrhotic rats. Norepinephrine significantly increased the activity of ROCK-1 in normal rats but not in cirrhotic ones. Norepinephrine significantly increased ROCK-1 activity in cirrhotic rats when GRK-2 inhibitor was used. CONCLUSIONS: β-arrestin-2 expression and its interaction with GPCRs are significantly upregulated in the mesenteric arteries in patients and rats with cirrhosis. These upregulations result in GPCR desensitization, G-protein dysfunction and ROCK inhibition. These may explain the decreased contractility of the mesenteric artery in response to vasoconstrictors.
